scholarly journals Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

2006 ◽  
Vol 73 (5) ◽  
pp. 1635-1645 ◽  
Author(s):  
G. Valsesia ◽  
G. Medaglia ◽  
M. Held ◽  
W. Minas ◽  
S. Panke
Keyword(s):  
Mass Spectrometry ◽  
Beneficial Effect ◽  
Specific Activity ◽  
Kanamycin Resistance ◽  
Leader Peptide ◽  
Kanamycin Resistance Gene ◽  
Gram Positive Bacteria ◽  
Tryptic Cleavage ◽  
Polypeptide Antibiotics ◽  
Staphylococcus Gallinarum

ABSTRACT Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.

Chicken Pituitary Glycoproteins: New Isolation Method

1999 ◽  
Vol 64 (9) ◽  
pp. 1510-1516
Author(s):  
Helena Ryšlavá ◽  
Jana Krešlová ◽  
Jana Barthová ◽  
Tomislav Barth
Keyword(s):  
Mass Spectrometry ◽  
Luteinizing Hormone ◽  
New Method ◽  
Hormonal Activity ◽  
Isolation Method ◽  
Molecular Weights ◽  
Testosterone Production ◽  
Maldi Tof ◽  
Tryptic Cleavage

A new method for isolation of glycoproteins from chicken pituitaries was applied. The procedure consist of chromatography on ConA-Sepharose and by HPLC on S Hyper D and Vydac C4 columns. The hormonal activity of the glycoproteins was tested by determining their stimulatory effect on cAMP or testosterone production. Molecular weights of the products of tryptic cleavage of the hormone were determined using mass spectrometry (MALDI TOF). A comparison of the values obtained with theory shows that the protein is the β-unit of chicken luteinizing hormone.

scholarly journals Reduced Virulence of a Bordetella bronchiseptica Siderophore Mutant in Neonatal Swine

2001 ◽  
Vol 69 (4) ◽  
pp. 2137-2143 ◽  
Author(s):  
Karen B. Register ◽  
Thomas F. Ducey ◽  
Susan L. Brockmeier ◽  
David W. Dyer
Keyword(s):  
Nasal Cavity ◽  
Resistance Gene ◽  
Parent Strain ◽  
Virulent Strain ◽  
Clinical Signs ◽  
Kanamycin Resistance ◽  
Live Vaccine ◽  
Bordetella Bronchiseptica ◽  
Kanamycin Resistance Gene ◽  
Phosphate Buffered Saline

ABSTRACT One means by which Bordetella bronchiseptica scavenges iron is through production of the siderophore alcaligin. A nonrevertible alcaligin mutant derived from the virulent strain 4609, designated DBB25, was constructed by insertion of a kanamycin resistance gene into alcA, one of the genes essential for alcaligin biosynthesis. The virulence of the alcA mutant in colostrum-deprived, caesarean-delivered piglets was compared with that of the parent strain in two experiments. At 1 week of age, piglets were inoculated with phosphate-buffered saline, 4609, or DBB25. Two piglets in each group were euthanatized on day 10 postinfection. The remainder were euthanatized at 21 days postinfection. Clinical signs, including fever, coughing, and sneezing, were present in both groups. Nasal washes performed 7, 14, and 21 days postinoculation demonstrated that strain DBB25 colonized the nasal cavity but did so at levels that were significantly less than those achieved by strain 4609. Analysis of colonization based on the number of CFU per gram of tissue recovered from the turbinate, trachea, and lung also demonstrated significant differences between DBB25 and 4609, at both day 10 and day 21 postinfection. Mild to moderate turbinate atrophy was apparent in pigs inoculated with strain 4609, while turbinates of those infected with strain DBB25 developed no or mild atrophy. We conclude from these results that siderophore production by B. bronchiseptica is not essential for colonization of swine but is required for maximal virulence. B. bronchiseptica mutants with nonrevertible defects in genes required for alcaligin synthesis may be candidates for evaluation as attenuated, live vaccine strains in conventionally reared pigs.

scholarly journals Insights into AMS/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Silvia C Bobeica ◽  
Shi-Hui Dong ◽  
Liujie Huo ◽  
Nuria Mazo ◽  
Martin I McLaughlin ◽  
...  
Keyword(s):  
Structural Data ◽  
Leader Peptide ◽  
Gram Negative Bacteria ◽  
Protease Domain ◽  
Peptide Recognition ◽  
Gram Positive Bacteria ◽  
Dual Functional ◽  
Signaling Peptides

The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.

scholarly journals Uptake of 2-Oxoglutarate inSynechococcus Strains Transformed with the Escherichia coli kgtP Gene

2000 ◽  
Vol 182 (1) ◽  
pp. 211-215 ◽  
Author(s):  
María Félix Vázquez-Bermúdez ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Escherichia Coli ◽  
Gene Cassette ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Gene ◽  
Gene Encoding ◽  
Resistance Gene Cassette ◽  
Low Levels ◽  
Active Transport Process ◽  
Taxonomic Groups ◽  
Pcc 7942

ABSTRACT A number of cyanobacteria from different taxonomic groups exhibited very low levels of uptake of 2-[U-14C]oxoglutarate.Synechococcus sp. strain PCC 7942 was transformed with DNA constructs carrying the Escherichia coli kgtP gene encoding a 2-oxoglutarate permease and a kanamycin resistance gene cassette. The Synechococcus sp. strains bearing thekgtP gene incorporated 2-oxoglutarate into the cells through an active transport process. About 75% of the radioactivity from the 2-[U-14C]oxoglutarate taken up that was recovered in soluble metabolites was found as glutamate and glutamine. 2-Oxoglutarate was, however, detrimental to the growth of aSynechococcus sp. strain bearing the kgtP gene.

Formaldehyde effects on kanamycin resistance gene of inactivated recombinant Escherichia coli vaccines

2020 ◽  
Vol 42 (11) ◽  
pp. 2223-2230
Author(s):  
Rafael A. Donassolo ◽  
Marcos Roberto A. Ferreira ◽  
Clóvis Moreira Jr ◽  
Lucas M. dos Santos ◽  
Emili Griep ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Gene ◽  
Kanamycin Resistance ◽  
Recombinant Escherichia Coli ◽  
Kanamycin Resistance Gene

Half-Life Determination of 41Ca and Some Other Radioisotopes

Radiocarbon ◽  
1992 ◽  
Vol 34 (3) ◽  
pp. 436-446 ◽  
Author(s):  
Walter Kutschera ◽  
Irshad Ahmad ◽  
Michael Paul
Keyword(s):  
Mass Spectrometry ◽  
Electron Capture ◽  
Half Life ◽  
Specific Activity ◽  
Low Energy ◽  
X Rays ◽  
Weighted Mean ◽  
Electron Capture Decay

We have performed a new determination of the half-life of 41Ca by measuring the specific activity of an enriched Ca material with known 41Ca abundance. We measured the activity via the 3.3-keV X-rays emitted in the electron capture decay of 41Ca, and the 41Ca abundance was measured by low-energy mass spectrometry. The result, t1/2 = (1.01 ± 0.10) × 105 yr, agrees with the recent ‘geological’ half-life of Klein et al., (1991), t1/2 = (1.03 ± 0.07) × 105 yr, and with the corrected value of Mabuchi et al. (1974), t1/2 = (1.13 ± 0.12) × 105 yr. We recommend the weighted mean of these three measurements, t1/2 = (1.04 ± 0.05) × 105 yr, as the most probable half-life of 41Ca. We also discuss the situation of the radioisotopes, 32Si, 44Ti, 79Se and 126Sn, whose half-lives, though still uncertain, are potentially interesting for future AMS studies and other applications.

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